Effect of water diuresis with hydrogen saturation on the course of acute kidney damage during the separation of oxidation and phosphorylation

Molecular hydrogen has the ability to penetrate cells, easily reach mitochondria, overcome body barriers, penetrate areas of ischemia, edema and inflammation, improve energy supply by supplying additional electrons and have antioxidant and anti-inflammatory effects by neutralizing highly reactive hydroxyl radical and peroxynitrite. In this experiment, we included 60 nonlinear male rats weighing 0.16–0.18 kg and investigated the effect of a negative redox potential solution -297.3±5.27 mV with a molecular hydrogen saturation of 1.2 ppm on the functional-biochemical processes of the kidneys in tissue hypoxia in moderately resistant rats during the separation of oxidation and phosphorylation with the introduction of 2,4-dinitrophenol at a dose of 3 mg/kg. All studies were performed on moderately stable rats. Experimental, functional, biochemical, enzyme-linked immunosorbent, physicochemical, histoenzymochemical, and statistical research methods were used. Under conditions of renal hypoxia in the separation of oxidation and phosphorylation, the use of a solution of negative redox reabsorption of sodium ions in the distal nephron reduces the manifestations of tubular proteinuria, increases the activity of succinate dehydrogenase in the proximal nephron and reduces the redox potential of urine to negative values. Negative redox potential solution with molecular hydrogen saturation has a protective effect on the kidneys and reduces elevated levels of proinflammatory cytokines of tumor necrosis factor-α, interleukin-1-β, and interleukin-6 in blood plasma, and causes oxidative modification of proteins in the renal cortex for their hypoxia in the separation of oxidation and phosphorylation.


INTRODUCTION
The main rule of kidney energy supply is that the bulk of oxygen absorbed by this organ is used to ensure the reabsorption of sodium ions [1][2][3]. According to this rule, the kidneys will be particularly responsive to disorders such as tissue hypoxia, which can occur due to the introduction of 2,4-dinitrophenol [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. In tissue hypoxia, the separation of oxidation and phosphorylation (SOP) creates conditions to activate lipid peroxidation, a significant energy deficit of the distal nephron [2,5]. Hypoxia in the renal tubules is not accompanied by significant inhibitions of the renal blood supply due to SOP. Under these conditions, a sufficient level of glomerular filtration, filtration fraction of sodium ions, and load on the energy-dependent mechanisms of the proximal and distal tubules are likely to be maintained. However, as energy is lacking, there will be significant reactions of proximal nephron damage and distal energy deficiency [1].
Thus, it is important to study the functional, biochemical, enzyme-linked immunosorbent, and histoenzymochemical changes of the kidneys under tissue hypoxia due to the SOP by introducing 2,4-dinitrophenol [15]. In order to correct the detected disorders, it is advisable to use an antioxidant solution of negative redox potential with hydrogen saturation, which has the ability to penetrate cells, easily reach mitochondria, and overcome body barriers: hemato-encephalic, hemato-thyroid, hemato-testicular, hemato-cochlear, hemato-ophthalmic, penetrate areas of ischemia, edema and inflammation, improve energy supply through the supply of additional electrons and exhibit antioxidant action by neutralizing highly reactive hydroxyl radical and peroxynitrite [4,6,8]. Considering the facts mentioned above, investigating the mechanisms of a negative redox potential solution with molecular hydrogen saturation on the functional and biochemical processes of the kidneys in tissue hypoxia is relevant and timely. The objective of the study was to determine the effect of a negative redox potential solution with molecular hydrogen saturation of 1.2 ppm on the functional and biochemical processes of the kidneys in tissue hypoxia in moderately resistant rats during SOP.

MATERIAL AND METHODS
The experiments were performed on 60 nonlinear male rats weighing 0.16-0.18 kg. Prior to the study, all experimental animals were adapted to a hyposodium diet and received food with a low content of sodium ions (wheat grain) and ordinary tap water for 3 days.
Resistance of rats to acute hypoxia was assessed by the time of loss of posture on the "high plateau" of acute hypobaric hypoxia and the time of general stay of animals from the moment of reaching "altitude" 12,000 m to the second agonal breath (lifetime or reserve time) and recovery time from the beginning of the descent. There were 3 groups of animals: high-, medium-, and low-resistant [1]. All studies were performed on moderately stable rats. Simulation of experimental tissue hypoxia was performed by a single intraperitoneal administration of 2,4-dinitrophenol at a dose of 3 mg/kg body weight [2]. The redox potential of water and urine (URP) was determined by an oxygen-reducing potential (ORP) -meter [13]. The effect of loading with ordinary tap water (redox potential 90.4±1.45 mV) the water of negative redox potential with hydrogen saturation 1.0-1.2 ppm and redox potential -297.3±5.27 mV was obtained using a new generation generator Blue Water 900 (Korea), mode 1, which contains an improved proton-exchange membrane PEM/SPE, being also a solid polymer electrolyte [9,11,14]. Figure. 1 Influence of water load of 5% of body weight with molecular hydrogen saturation 1.2 ppm on diuresis, concentration of potassium ions in urine, redox potential of urine and urinary protein excretion under hypo sodium diet with registration of urine collection for 2 hours during SOP after administration of 2,4-dinitrophenol at a dose of 3 mg kg. Note: here and in fig. 2 and  3. Control (C) -intact animals with a load of ordinary tap water, SOP -SOP when loaded with ordinary tap water, SOP + H2 -SOP when loaded with water with molecular hydrogen saturation 1.2 ppm. Significance of differences was noted in comparison with C: *-p < 0,05; **-p < 0,02; ***-p < 0,01; ****-p < 0,001 compared to the SOP: • -p < 0,05; •• -p < 0,02; ••• -p < 0,01. . Control (C) -intact animals with a load of ordinary tap water, SOP -SOP when loaded with ordinary tap water, SOP + H2 -SOP when loaded with water with molecular hydrogen saturation 1.2 ppm. Significance of differences was noted in comparison with C: *-p < 0,05; **-p < 0,02; ***-p < 0,01; ****-p < 0,001 compared to the SOP: . Control (C) -intact animals with a load of ordinary tap water, SOP -SOP when loaded with ordinary tap water, SOP + H2 -SOP when loaded with water with molecular hydrogen saturation 1.2 ppm. Significance of differences was noted in comparison with C: *-p < 0,05; **-p < 0,02; ***-p < 0,01; ****-p < 0,001 compared to the SOP: The functional state of the kidneys was studied under a water load. Rats were injected intragastrical with a metal probe using ordinary tap water, water of negative redox potential with additional molecular hydrogen saturation, heated at a temperature of 37℃ in the amount of 5% of body weight. The diuresis (V) amount was estimated in ml/2h × 100 g body weight. After obtaining plasma using water loading, the euthanasia of animals was performed by decapitation under light ether anesthesia, and blood was collected in tubes with heparin. In plasma and urine, the creatinine concentration was determined by reaction with picric acid, sodium and potassium ions -using flame photometry, and protein in urine by the sulfosalicylic method. The excretion of sodium ions (ENa + ) and protein (E pr ) was evaluated by the formulas: ENa + =V×UNa + ; E pr =V×U pr , where UNa + and U pr are the concentrations of sodium ions and protein in the urine, respectively. The clearance of sodium-free (CH 2 ONa + ) water was calculated by the formula: CH 2 ONa + =V-V×UNa + /PNa + , where PNa + is the concentration of sodium ions in blood plasma. Reabsorption of sodium ions in the distal nephron (T d Na + ) was evaluated by the formula: T d Na + =(PNa + -UNa + )×V [7]. Concentrations of sodium and potassium ions in urine (UK+) were determined by flame photometry [3].
The activity of succinate dehydrogenase in the proximal nephron ( p SDA) of the kidneys was determined by histoenzymochemical method on cryostat sections 10 μm thick using 0.2 M phosphate buffer (pH 7.6), 0.2 M sodium succinate solution and nitrosine tetrazolium. Quantitative analysis of the activity of the studied enzyme was performed by the method of spot test ac-cording to Avtandilov G.G. by superimposing templates on the projection of the drug with the subsequent calculation of test points [3]. Enzyme-linked immunosorbent assays were performed using standard kits Amersham (England) and Immuno Nuclear Corporation (USA) to determine the concentration of tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6). The content of oxidatively modified proteins in the renal cortex (OMP c ) was determined by reaction with 2,4-dinitrophenylhydrazine to form hydrazones with a characteristic absorption spectrum. The degree of OMP c was judged by the number of formed aldehyde and ketone groups [1]. The content of diene conjugates (DC c ) and malonic aldehyde (MA c ) was determined in the renal cortex [3]. According to the statistical analysis of the obtained results, which corresponded to the normal distribution, the methods of variational statistics generally accepted in medicine were used, and the following were calculated: arithmetic mean sample (x) and arithmetic mean standard error (Sx). When estimating the probability of the difference between the average values, the coefficient t was calculated. Reliability (probability of zero hypothesis) for a given t and the number of degrees of freedom was calculated according to the Student's method in a two-way test, and strength (according to an alternative hypothesis) was determined by a one-sided test.
To claim the probability of the difference, the standard level of probability p<0.05, generally accepted in bio-biological studies, was considered. We agreed on the use of the Mann-Whitney test. Mann-Whitney (U-test) is a non-parametric statistical test used to assess the difference between two samples in the level of any

RESULTS
Hypoxia of the kidneys during SOP was accompanied by inhibition of urinary excretion. An increase in the concentration of potassium ions in the urine and protein excretion was detected. The URP did not change. The use of negative redox fluid with molecular hydrogen saturation increased the level of diuresis, decreased the concentration of potassium ions in the urine, protein excretion, and inhibited the negative redox potential of urine ( Figure 1). Under conditions of renal hypoxia due to SOP, there was an increase in excretion of sodium ions, inhibition of clearance of sodium ion-free water, decreased T d Na + and inhibition of р SDA. Using a negative redox fluid with molecular hydrogen saturation caused a decrease in the loss of sodium ions in the urine, increased clearance of sodium ion-free water, and increased T d Na + , р SDA ( Figure 2).
Under conditions of renal hypoxia due to the SOP, the systemic low-intensity inflammation was activated, as indicated by an increase in plasma concentrations of proinflammatory cytokines: TNFα, IL-1β, IL-6. In the kidneys under these conditions, there was an increase in OMP c , MA c by 68.9% (p<0.02), and DC c by 50% (p<0.02). The use of a solution of negative redox potential with molecular hydrogen saturation showed a normalizing anti-inflammatory effect on the state of cytokines: TNFα, IL-1β, and IL-6 ( Figure 3). OMP c decreased according to the application of a solution of negative redox potential with molecular hydrogen saturation and also the level of MA c by 21.6% (p<0.05) and DC c by 26.7% (p<0.05).

DISCUSSION
The introduction of 2,4-dinitrophenol decreased ATP levels in the renal tubules due to the SOP in violation of the main energy-dependent process of the renal tubules -reabsorption of sodium ions, which led to the development of cation loss syndrome. This is confirmed by an increase in the excretion of sodium ions and a decrease in the clearance of sodium-free water. Damage to the intestinal and liver barriers on the background of energy deficiency led to the translocation of endotoxin from the intestinal lumen into the blood, which led to an increase in the concentration of TNFα, which in turn caused increased cytokines IL-1β and IL-6.
Due to their pro-inflammatory properties, cytokines generated active radicals and additional damage reactions to the renal tubules [16][17][18][19][20][21][22][23][24] with the increasing manifestation of the syndrome of loss of sodium ions. The points of influence of molecular hydrogen concerning the rupture of a large vicious circle for SOP in rats moderately resistant to hypoxia are based on the results of other studies [25][26][27][28][29][30] (Figure 4).  SOP leads to growth in the plasma of proinflammatory cytokines of TNFα, IL-1β, and IL-6 (anti-inflammatory effect molecular hydrogen [10,16]) and causes inhibition of substantially energy-dependent distal reabsorption of sodium ions and development of proximal tubular proteinuria with increasing protein excretion. Furthermore, it leads to urinary sodium ion loss syndrome with activation of the intrarenal renin-angiotensin-aldosterone system (RAAS) with decreased V leading to an increase in the concentration of potassium ions in the urine due to the kaliuretic effect of aldosterone and an increase in the proximal tubule damage with inhibition of р SDA (energy protection effect molecular hydrogen [30]). This leads to further hypoxic activation of lipid peroxidation and OMP c , with increasing MA c and DC c (antioxidant effect molecular hydrogen [25][26][27][28][29][30]) increasing damage to the proximal tubule and the pathogenic effects on the kidneys during SOP. In addition, molecular hydrogen provided the rupture of small vicious circles by the SOP.
Urinary sodium ion loss syndrome with activation of the intrarenal renin-angiotensin-aldosterone system and decreased diuresis led to an increase in the concentration of potassium ions in the urine due to the kaliuretic effect of aldosterone. In addition, it increased damage to the proximal tubule with inhibition of succinate dehydrogenase activity (rupture of the first small vicious circles with molecular hydrogen) [28][29][30], increasing the syndrome of loss of sodium ions in the urine.
The increase in the concentration of potassium ions in urine due to the kaliuretic effect of aldosterone and the increase in damage to the proximal tubule with inhibition of succinate dehydrogenase activity (rupture of the second small vicious circles due to molecular hydrogen) [25][26][27] leads to further hypoxic activation of lipid peroxidation in the content of diene conjugates. Furthermore, the increase in the content of malonic aldehyde in the renal cortex leads to further damage to the proximal tubule with inhibition of succinate dehydrogenase activity.
Hypoxic activation of lipid peroxidation and oxidative modification of proteins in the renal cortex with increasing content of diene conjugates and malonic aldehyde in the renal cortex (rupture of the third small vicious circles due to the antioxidant effect of molecular hydrogen) [28] increases damage to the proximal tubule and leads to lipid peroxidation and oxidative modification of proteins in the cortical substance of the kidneys with increasing content of diene conjugates and malonic aldehyde.

CONCLUSION
Under conditions of renal hypoxia due to SOP, the use of a solution of negative redox potential with molecular hydrogen saturation improves the condition of the distal tubule, reduces the manifestations of proteinuria in the nephron, and reduces the concentration of potassium ions in the urine against the background of reducing the redox potential of urine to negative values. The use of a solution of negative redox potential with molecular hydrogen saturation has a protective effect on the kidneys due to its antioxidant, anti-inflammatory, and nephroprotective properties. It reduces elevated levels of proinflammatory cytokines TNFα, IL-1β, and IL-6 in the blood and oxidative modification of proteins in the cortical substance of the kidneys in their hypoxia due to the SOP.